We wish to determine how isoniazid inhibits the synthesis of mycolic acids in Mycobacterium tuberculosis H37Ra and whether this inhibition is the primary mode of action of the drug. We are also studying how mycolic acids are synthesized. We developed a high performance liquid chromatographic method to fractionate the complex mixtures of C30 to C56 fatty acids from M. tuberculosis H37Ra. Silica column is used to achieve structural class separation and reverse-phase column is used to achieve separation according to chain length. The structures of these fatty acids (which are potential mycolic acid precursors) will be determined. We are developing a cell-free system that is capable of synthesizing a mycolic acid precursor called mero-mycolic acid (C50 to C56). Our crude cell-free system is sensitive to isoniazid.